Optimizing oligonucleotide libraries for mutagenesis

Oligonucleotide libraries are commonly used for mutagenesis of proteins that can be screened for potential improvements in biological function, such as enhanced expression levels, stability, solubility, enzymatic activity, or binding affinity. While you can create libraries of sequences by simply having random bases incorporated into synthetic oligonucleotides, this method introduces stop codons and a higher proportion of certain amino acids, which increases the time and resources required to screen the resulting libraries. Alternatively, using the 3 degenerate bases NNK will reduce, but not eliminate the number of possible stop codons and improves proportional codon biases, but will not eliminate, the number of possible stop codons and codon biases.

These issues can be avoided by incorporating codon trimers into your oligos. IDT trimer mixes do not contain stop codons, and trimer mixes provide a balanced mix of the amino acid codons, resulting in equal representation of each unique codon.

Trimer codon mix options for balanced amino acid coding

Trimer 19 contains an equal ratio of the following 19 codons (excluding CYS):

AAA, AAC, ACT, ATC, ATG

CAG, CAT, CCG, CGT, CTG

GAA, GAC, GCT, GGT, GTT

TAC, TCT, TGG, TTC

Trimer 20 contains an equal ratio of the following 20 codons (including CYS):

AAA, AAC, ACT, ATC, ATG

CAG, CAT, CCG, CGT, CTG

GAA, GAC, GCT, GGT, GTT

TAC, TCT, TGC, TGG, TTC

To limit synthesis complexity and facilitate fast delivery, we restrict the total length of oligos with trimer insertions to 100 total bases (counting each trimer as 3 bases). The number of codon trimer insertions within an oligonucleotide is also limited to 15. However, we are able to consider other designs outside of these specifications as non-catalog requests. Just contact us at noncat@idtdna.com.

Create your own custom trimer codon mixes

We can also incorporate a custom trimer codon into oligos. This allows you to customize the ratios of trimers incorporated. To order a custom trimer codon mix, email noncat@idtdna.com with your name, organization, and sequence designs. Oligos containing Custom Trimer Mixes can be ordered as standard desalted oligos (recommended for oligos containing mixes), provided that other modifications included in the sequences do not require HPLC.